The radical SAM protein HemW is a heme chaperone

HAL Id: hal-01915476

https://hal-amu.archives-ouvertes.fr/hal-01915476

Submitted on 9 Nov 2018

HAL

is a multi-disciplinary open access archive for the deposit and dissemination of sci- entific research documents, whether they are pub- lished or not. The documents may come from teaching and research institutions in France or abroad, or from public or private research centers.

L’archive ouverte pluridisciplinaire

destinée au dépôt et à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d’enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Distributed under a Creative Commons

Vera Haskamp, Simone Karrie, Toni Mingers, Stefan Barthels, François Alberge, Axel Magalon, Katrin Müller, Eckhard Bill, Wolfgang Lubitz, Kirstin

Kleeberg, et al.

To cite this version:

Vera Haskamp, Simone Karrie, Toni Mingers, Stefan Barthels, François Alberge, et al.. The radical SAM protein HemW is a heme chaperone. Journal of Biological Chemistry, American Society for Biochemistry and Molecular Biology, 2018, 293 (7), pp.2558 - 2572. �10.1074/jbc.RA117.000229�.

�hal-01915476�

1 The radical SAM protein HemW is a Heme Chaperone

Vera Haskamp

, Simone Karrie

, Toni Mingers

, Stefan Barthels

, François Alberge

, Axel Magalon

, Katrin Müller

, Eckhard Bill

, Wolfgang Lubitz

, Kirstin Kleeberg

, Peter Schweyen

, Martin Bröring

, Martina Jahn

and Dieter Jahn

1

Institute of Microbiology, University Braunschweig, Braunschweig, Germany,

LCB, CNRS, Aix- Marseille Université, Marseille, France,

Max Planck Institute for Chemical Energy Conversion, Mülheim an der Ruhr, Germany,

Institute of Inorganic and Analytical Chemistry, University Braunschweig, Braunschweig, Germany,

Braunschweig Centre of Integrated Systems Biology (BRICS), University Braunschweig, Braunschweig, Germany

Running title: Novel Heme Chaperone HemW

To whom correspondence should be addressed: Dieter Jahn, Braunschweig Centre of Integrated Systems Biology (BRICS), University Braunschweig, Rebenring 56, D-38106 Braunschweig, Germany. Tel.: +4953139155101, E-mail: d.jahn@tu-bs.de

Keywords: HemW, heme chaperone, radical SAM, iron-sulfur cluster, Escherichia coli

ABSTRACT

Radical S-adenosylmethionine (SAM) enzymes exist in organisms from all kingdoms of life, and all of these proteins generate an adenosyl radical via the homolytic cleavage of the S–C(5’) bond of SAM. Of particular interest are radical SAM enzymes, such as heme chaperones, that insert heme into respiratory enzymes.

For example, heme chaperones insert heme into target proteins, but have been studied only for the formation of cytochrome c type hemoproteins. Here, we report that a radical SAM protein, the heme chaperone HemW from bacteria, is required for the insertion of heme b into respiratory chain enzymes. As other radical SAM proteins, HemW contains three cysteines and one SAM coordinating an [4Fe-4S] cluster, and we observed one heme per subunit of HemW. We found that an intact iron-sulfur cluster was required for HemW dimerization and HemW-catalyzed heme transfer, but not for stable heme binding. A

bacterial two-hybrid system screen identified bacterioferritins and the heme- containing subunit NarI of the respiratory nitrate reductase NarGHI as proteins that interact with HemW. We also noted that the bacterioferritins potentially serve as heme donors for HemW. Of note, heme that was covalently bound to HemW was actively transferred to a heme-depleted, catalytically inactive nitrate reductase, restoring its nitrate-reducing enzyme activity. Finally, the human HemW orthologue radical SAM domain–

containing 1 (RSAD1) stably bound heme.

In conclusion, our findings indicate that the radical SAM protein family HemW/RSAD1 is a heme chaperone catalyzing the insertion of heme into hemoproteins.

__________________________________

Radical SAM enzymes have been

discovered in organisms from all kingdoms

of life (1-3). The currently known 114.000

2 radical SAM proteins catalyze a broad

variety of challenging chemical reactions (4-7). For instance, humans possess eight radical SAM proteins: 1. MOCS1 involved in molybdenum cofactor biosynthesis, 2. LIAS for the formation of lipoic acid, 3. CDK5RAP for 12-methylthio-N(5)- isopentenyladenosine synthesis, 4. CDKAL1 required for methylthio-N(6)- threonylcarbamoyladenosine formation, 5. TYW1 for wybutosine biosynthesis, 6. ELP3 for 5-methoxycarbonymethyl uridine, 7. Viperin and 8. RSAD1, respectively (reviewed in (2). Viperin is involved in the innate antiviral response (8). However, the exact enzymatic function of human viperin and RSAD1 are currently unknown.

All have in common the generation of a adenosyl radical via the homolytic cleavage of the S–C(5’) bond of SAM.

SAM and three cysteine residues generally coordinate a [4Fe-4S] cluster, leading to the typical CX

CX

C protein sequence signature of radical SAM enzymes (1). The first crystal structure of a radical SAM enzyme was solved for an enzyme of bacterial heme biosynthesis called coproporphyrinogen III dehydrogenase (HemN) (9). Three iron atoms of the [4Fe-4S] cluster of HemN are coordinated by the three cysteine residues Cys

, Cys

and Cys

of the conserved motif (9,10). A fourth cysteine (Cys

) is not essential for [4Fe-4S] cluster coordination, but for catalysis (10). During the catalytic reaction for the conversion of coproporphyrinogen III into protoporphyrinogen IX the [4Fe-4S]

cluster first gets reduced. This leads to the homolytic cleavage of the SAM S-C(5’) bond and the formation of a 5’-deoxyadenosyl radical. The generated radical then removes stereospecifically one hydrogen atom from a propionate side chain of the substrate to yield 5’-deoxyadenosine and a substrate radical which in turn leads to the desired decarboxylation reaction (9,11). However, the presence of HemN proteins (also

named CPDH) carrying the CX

CX

CXC motif is limited to a few classes of bacteria (12). Multiple hemN-like genes encoding proteins of significant amino acid sequence homology were found in most classes of organisms with the exception of fungi and were originally annotated as coproporphyrinogen III oxidase.

Recently, the corresponding Lactococccus lactis protein was observed to bind heme and considered to play a role in maturation of the cytochrome oxidoreductase of the bacterium. It was therefore renamed HemW (13). L. lactis HemW (NP_267295.1) displays high homology to Escherichia coli coproporphyrinogen III dehydrogenase HemN (50 % amino acid sequence similarity). Surprisingly, L. lactis HemW did not show CPDH activity in vitro and in vivo (13). In contrast to E. coli HemN, L. lactis HemW is missing 47 N-terminal amino acids and the fourth cysteine residue of the conserved CX

CX

CXC motif (13). E. coli possesses HemN and additionally a HemW-like protein annotated as YggW (NP_417430.1), a protein of hypothetical function. Because of the high degree of amino acid sequence identity of 36% (58%

homology) to L. lactis HemW we renamed YggW to HemW in the present work.

Relatedly, the corresponding Pseudomonas aeruginosa protein (WP_003128950) with an amino acid sequence identity of 31%

(50% homology) to L. lactis HemW was also renamed to HemW. Similar to L. lactis HemW, the HemWs of E. coli and P. aeruginosa displayed a truncated N-terminus and the conserved cysteine motif lacking the fourth cysteine. A corresponding amino acid sequence alignment is shown in figure S1.

The only well characterized systems for

the insertion of heme into proteins are the

different cytochrome c biogenesis machine

ries (14). Cytochrome c is involved in

multiple electron transport chains. For

cytochrome c formation rotoheme IX and

the apocytochrome are transported through

3 the membranes of prokaryotes,

mitochondria and chloroplasts.

Subsequently, a covalent thioether bond is actively formed between at least one cysteine and a vinyl group of the heme.

Currently, 5 different systems are proposed to perform the processes of heme insertion into a c-type cytochrome which differ in their level of complexity and are found in distinct organisms (14). Sporadically, reports on other heme binding and potential heme inserting proteins occur in the literature, as for NikA, an E. coli periplasmic nickel-protein (15)or human glyceraldehyde-3-phosphate

dehydrogenase (16). Furthermore, a putative role for the protein Surf1 of Paracoccus denitrificans as a heme a chaperone involved in COX biogenesis was described (17). Recently, we described the heme binding protein HemW from Lactococcus lactis hypothesizing that HemW is involved in heme trafficking (13).

Here, we provide biochemical, genetic and biophysical evidences that the bacterial HemW proteins are heme chaperones for the insertion of heme b into enzymes of respiratory chains.

Results

E. coli HemW has no coproporphyrinogen III dehydrogenase activity in vitro and in vivo - E. coli HemN and HemW amino acid sequences are 33% identical but differ in two major features. E. coli HemN carries extra 46 N-terminal amino acid residues which have been proposed to be crucial for substrate binding (9). Moreover, the fourth cysteine of the HemN CX

CX

CXC motif is replaced by a phenylalanine in HemW. These differences are found in all HemW-like proteins (13).

To investigate whether HemW carries coproporphyrinogen III dehydrogenase (CPDH) activity, it was first analyzed in vitro. For this purpose E. coli HemN and HemW were recombinantly produced and purified to apparent homogeneity. In

contrast to HemN (18), E. coli HemW completely failed to catalyze the conversion of coproporphyrinogen III into protoporphorynogen IX. In a next step in vivo complementation experiments using the E. coli ∆hemN strain JKW3838 under anaerobic growth conditions were performed. Due to the presence of the oxygen-dependent coproporphyrinogen III oxidase HemF this mutant grew efficiently under aerobic conditions (data not shown).

However, in the absence of oxygen a severe growth impairment was detected.

Minimal remaining growth might result from fermentative energy generation or residual HemF activity. Clearly, E. coli hemN (pET3-hemN) complemented the ∆hemN E. coli strain to wildtype

comparable growth, while

E. coli pGEXhemW failed to restore anaerobic growth of the mutant (Fig.1).

Both results clearly indicate that E. coli

HemW does not harbor

coproporphyrinogen III dehydrogenase

activity as was also observed for L. lactis

HemW (13).These results are in agreement

with heme auxotrophy reported for a

Salmonella typhimurium hemF/hemN

double mutant carrying an intact

hemW(19). In order to test if the deviating

N-terminus and the missing fourth cysteine

residue were responsible for the observed

behavior we constructed a HemW F25C

protein carrying the fourth cysteine and a

HemW-HemN hybrid protein carrying the

46 N-terminal amino acids of HemN fused

to HemW F25C. Nevertheless,

HemWF25C+46N-term did not show any

coproporphyrinogen III dehydrogenase

activity in vitro. In agreement, no

complementation of the E. coli ∆hemN

strain under anaerobic conditions was

observed with any other of the HemW

variants. Obviously, additional structural

elements are required for efficient HemN

activity. Consequently, HemW is not an

inactivated potential coproporphyrinogen

III dehydrogenase.

4 E. coli HemW binds a [4Fe-4S] cluster -

For the biochemical and biophysical characterization, E. coli HemW was produced as glutathione S-transferase (GST) fusion protein in E. coli BL21 DE3.

After anaerobic chromatographic purification and removal of the GST tag by PreScission protease cleavage, an apparent homogenous protein was obtained. SDS- PAGE analysis revealed a single protein band after staining with Coomassie Blue (Fig. 2A). The protein had a relative molecular mass of ̴ 45,000 ±5,000 which nicely corresponds to the calculated molecular mass for the HemW monomer of 42,584 Da. Approximately 12.5 mg of purified HemW were obtained per liter of culture. To elucidate if E. coli HemW coordinates an iron-sulfur cluster, the iron and sulfur contents of the protein were determined. For native, purified HemW, no obvious absorption around 410 - 425 nm was detectable in the UV/Vis absorption spectrum. Purified HemW exhibited 0.5 mol iron/0 mol sulfur per mol HemW and an A

:A

ratio of 0.04.

Consequently, we decided for a reconstitution of the obviously labile [Fe-S] cluster via treatment of the protein with iron ammonium citrate and lithium sulfide. After reconstitution the iron and sulfur content of HemW increased to 3.8 mol iron/2.5 mol sulfur per mol HemW and an A

:A

ratio of 0.19. As a consequence, the typical absorbance for [Fe-S] clusters at 420 nm became clearly visible (Fig. 2B). To further characterize the cluster type of HemW, the iron-sulfur cluster of HemW was reconstituted with

57

Fe-ammonium ferric citrate and Mössbauer spectroscopy of

Fe reconstituted HemW was performed.

Mössbauer spectra were recorded for

samples containing HemW. Spectra without further addition revealed one dominant quadrupole doublet (83% of the total intensity) with an isomer shift (δ) of 0.49 mm/s and a quadrupole splitting parameter (ΔE

) of 1.00 mm/s, which are typical of [4Fe-4S]

clusters (Fig. 2C, dashed line). Moreover, a second quadrupole doublet (17% of the total intensity) with an isomer shift (δ) of 1.48 mm/s and a quadrupole splitting parameter (ΔEQ) of 3.30 mm/s was detected (Fig. 2C, dotted line). The solid line in Figure 2 represents the superposition of the two quadrupole doublets. This spectrum is consistent with a coordination of the iron-sulfur cluster by three cysteine ligands and one potential N/O ligand. The three cysteine residues are likely the Cys

, Cys

and Cys

of the CX

CX

C motif at the N-terminus of the protein sequence. The high isomer shift of the second quadrupole doublet excludes an orgin from [Fe-S] clusters, but reveals high-spin Fe(II) sites with six hard O- or N- ligands; the component is therefore assigned to adventitiously bound Fe(II) in the protein, presumably remaining from the reconstitution procedure. However, various attempts to reduce the [4Fe-4S]

cluster with different electron donor systems (e.g. sodium dithionite, titanium III citrate, with redox mediators) for subsequent EPR analysis failed. In contrast, the [4Fe-4S] cluster of the related E. coli HemN could be reduced at such conditions (18) and employed for successful EPR measurements.

Surprisingly, cyclic voltammetry

measurements clearly indicated a redox

transition of the iron-sulfur cluster of

HemW at around -410 mV (Fig. 3). The

potential of -410 mV is in the range of

5 values found for other radical SAM

enzymes (20). At this redox potential both dithionite and titanium III citrate should serve as efficient electron donors for HemW. Obviously, electron donor compounds are prevented to access the [4Fe-4S] cluster for reduction, consequently no radical reaction can be initiated. A similar explanation has been suggested for the [Fe-S] cluster in succinate dehydrogenase subunit B, which appears to be inaccessible for oxidants and toxins (21).

The HemW [4Fe-4S]

cluster promotes protein dimerization - To study the influence of the iron-sulfur cluster on the oligomerization state of HemW, experiments using size-exclusion chromatography of anaerobically purified and reconstituted HemW and of a HemW variant (C16S-C20S-C23S) lacking the [4Fe-4S] cluster were performed (Fig. 4).

For the [4Fe-4S] cluster containing HemW, two fractions corresponding to monomeric (fraction 16) and dimeric protein (fraction 14) were detected (Fig. 4A). Calibration of the column revealed, that fraction 16 for the monomeric protein corresponded to a M

of 45,000 ± 5000 and fraction 14 for the dimeric protein to a Mr of 87,000 ± 6000.

Interestingly, an increased amount of iron- sulfur cluster was spectroscopically detected at 420 nm for the dimeric HemW species compared to the monomeric form (Fig. 4A, dashed line). Nevertheless, iron- sulfur clusters were also detected in monomeric HemW, which indicated a dynamic transition between monomeric and dimeric HemW. This transition between monomeric and dimeric proteins was tested by re-chromatography of the collected separated monomeric and dimeric HemWs in fraction 14 and fraction 16 on the gelfiltration column. We observed, that re-chromatography of the

dimeric HemW in fraction 14 resulted again in two equal sized protein absorption peaks in fractions 14 and 16 (Fig. 4C).

However, most iron sulfur cluster absorption was detected for the dimeric protein in fraction 14. Analogously, re- chromatography of monomeric protein in fraction 16 generated also two absorption maxima in fraction 14 and 16, however, with the bigger peak in fraction 16 representing the monomeric protein (Fig 4D). An analytical gelfiltration analysis of HemW without [Fe-S] cluster, caused by the replacement of cysteines 16, 20 and 23 to serines of the iron-sulfur cluster binding motif revealed only one single peak in fraction 16 corresponding to a monomeric protein (Fig. 4B). The C16S- C20S-C23S HemW variant was subjected to iron-sulfur cluster reconstitution experiment analogously to the wildtype protein prior these experiments, but remained iron-sulfur cluster free as determined spectroscopically and by iron and sulfur determinations. Obviously, an equilibrium exists between the monomeric and dimeric protein, however, formation of a dimeric HemW is favored by the incorporation of the iron-sulfur cluster.

E. coli HemW binds SAM - The amino acid

sequence analysis of E. coli HemW clearly

revealed two binding sites for S-denosyl-L-

methionine (SAM) similar to the radical

SAM enzyme HemN from E. coli. Overall,

in E. coli HemN 19 amino acid residues

are known from the crystal structure of the

protein to coordinate 2 SAM molecules

and 1 [4Fe-4S] cluster. Of the involved 19

HemN amino residues 11 (R184, G113,

T114, C66, C62, C69, Q172, D209, Y56,

G112, E145, clockweise around the

binding site (Fig. S2) were found identical

in HemW (R138, G67, T68, C20, C16,

C23, Q126, D163, Y10, G66, E95) and 3

homologous (I211 in HemN– M165 in

HemW, F240 – Y194, F68 – Y22), when

the amino acid sequences of the proteins

were aligned. Due to the low amino acid

6 sequence conservation at the C-terminus of

both proteins, an aligment of 4 amino acid residues of HemN (A243, A242, F310, I329) did not match the corresponding residues of HemW. The region of HemN is involved in coproporphyrinogen III coordination and represents most likely the heme binding region of HemW. Only the cysteine (C71) and the residue aside (G70) used to clearly differentiate HemN from HemW proteins were found clear cut different (F25, D24). For experimentally studying SAM-binding of HemW, the purified reconstituted protein was incubated with

C-SAM and the mixture passed over a desalting column for the removal of non-incorporated free SAM.

Protein-bound

C-SAM was subsequently quantified using liquid scintillation counting. The control experiment was carried out using BSA and

C-SAM. The HemW-

C-SAM complex was eluted in the protein-containing fractions (Fig. 5A, solid line, fractions 1-4). Some free

14

C-SAM eluted in the later, small molecule fractions. In contrast all

14

C-SAM incubated with BSA eluted in the small molecules fraction (Fig. 5A, dashed line, fractions 6-14). Consequently, SAM binding to HemW was clearly demonstrated. The highly conserved structure of the SAM and [4Fe-4S]-cluster binding site suggested the presence of two SAM molecules. However, due to the unknown amount of already bound SAM in the tested HemW proteins and the unknown exchange rate between bound and unbound SAM, it was not possible to determine the stoichiometry of SAM binding to HemW.

Analysis of the SAM cleavage capacity of HemW - The classical radical SAM enzyme chemistry requires the reduction of the [4Fe-4S] cluster, homolytic cleavage of the S–C(5’) bond of SAM with the generation of the 5’-deoxyadensosyl radical. HemN usually requires its substrate coproporphyrinogen III for

radical formation (10). However, in the absence of the substrate residual enzymatic SAM cleavage of usally less than 15% of the reaction without substrate was observed. To test HemW for full or residual SAM cleavage activity reconstituted HemW protein was incubated with SAM and with and without heme as potential substrate under reducing conditions. The disappearance of SAM with the parallel formation of deoxyadenosine was monitored by HPLC analysis (Fig 5B, solid line). The same experiment was performed as control with purified HemN in absence and presence of substrate (Fig. 5B, dashed and dotted line).

In this case under tested conditions E. coli HemN revealed full SAM cleavage activity in the presence of the substrate coproporphyrinogen III (Fig. 5B, dotted line) and less than 5% of its SAM cleavage activity without substrate (Fig. 5B, dashed line). Comparable residual SAM cleavage capacity was observed for HemW with the addition (Fig. 5B, solid line) and without the addition of heme (not shown). Clearly, E. coli HemW revealed only the residual SAM cleavage activity comparable to HemN without substrate (Fig. 5B).

HemW is a heme binding protein - Previous studies with the HemW homolog from L. lactis revealed heme binding to the protein (13). In order to test for heme binding of E. coli HemW and determine its specificity, the purified HemW protein and heme were incubated anaerobically

overnight and analyzed

spectrophotometrically (Fig. 6A, dotted line). As control the employed protein solution and free heme were analyzed in parallel. HemW showed only the typical protein absorption at 280 nm and little absorption for the iron-sulfur cluster (Fig 6A, solid line). Free heme showed the typical spectrum with peaks around 400 nm and 580 nm (Fig 6A, dashed line).

The HemW-heme complex revealed a

broad absorption peak between 380 and

7 420 nm besides the protein absorbance at

280 nm (Fig 6A, dotted line). In order to determine the specificity of heme binding, all three samples (HemW, heme, HemW- heme complex) were subjected to extensive dialysis overnight and subsequent spectroscopic analyses. While the spectrum for HemW did not change (Fig 6B, solid line) and the spectrum for the HemW-heme complex only lost its little increase around 400 nm (Fig 6B, dotted line), all free heme was gone (Fig 6B, dashed line). Identical results were obtained for the dialyzed and Superdex 200 gelfiltrated HemW-heme complex (data not shown). Interestingly, reduction ot the HemW-heme complex resulted in an increase of absorption at 424 nm generating a Soret band and further absorption peaks at 531 nm and 559 nm (Fig 6c, dashed line). In contrast, free reduced heme shows an absorption peak at around 400 nm. These results demonstrate the specificity of HemW-heme interaction.

For the further analysis of the nature and stoichiometry of HemW-heme interaction, complexes were analyzed via SDS PAGE with heme staining and acidified butanone extraction. HemW and equimolar amounts of heme were incubated overnight and subjected in duplicate to SDS PAGE analyses. Subsequently, one half of the gel was stained with Coomassie Brillant Blue for detection of separated proteins (Fig.

7A, lane 1), while the proteins on the second half of the gel were blotted onto a nitrocellulose membrane for heme staining.

The detection of the HemW bound heme was based on its intrinsic peroxidase activity by incubation with the ECL reagent (Fig. 7A, lane 2). The observed heme staining of E. coli HemW indicated stably bound heme. In order to obtain further evidence for the possible covalent nature of heme binding, butanone extraction experiments were performed which can result either in release of non- covalently linked heme in the organic phase or still bound, covalently linked

heme in the aqueous phase (22).

Cytochrome c was used as a positive control clearly indicating the presence of covalently bound heme in the aqueous phase (Fig. 7B, middle picture).

Hemoglobin with non-covalently bound heme was used as negative control. Here, almost all heme was extracted in the upper organic phase (Fig. 7B, right picture).

Butanone extraction of HemW incubated with heme revealed a completely clear upper phase and a slightly brownish lower phase (Fig. 7B, left picture). The presence of HemW derived heme in the lower aqueous phase indicated covalently bound heme. Even though our experiment pointed towards covalently bound heme, strong binding of the heme in a tight hydrophobic pocket resistant to SDS and butanone treatment can not be excluded.

Subsequently, the heme staining assay was used to identify the binding stoichiometry of HemW and heme. For this purpose, a solution of 10 µM HemW (Fig 7C, lanes 1 to 5) was titrated with heme in increasing amounts from 5 µM concentration to 25 µM (Fig 7C, lanes 6 to 10). The subsequent heme staining revealed an increase of the heme bound to HemW up to a heme concentration of 10 µM indicating that apparent saturation of the signal occurred after addition of equimolar amounts of heme (Fig 7C, lanes 6 to 10).

Alternatively, heme binding stoichiometry

by HemW was determined

spectroscopically by using 20 µM native HemW which was titrated with increasing amounts of heme. Measurements of the optical density at 416 nm revealed a heme- binding saturation at 20 µM (Fig. 7D).

These results clearly indicate a specific binding with a stoichiometry of one molecule heme per HemW monomer.

Heme binding is independent of the

presence of the iron-sulfur cluster -

Aerobically prepared HemW without iron-

sulfur cluster was binding heme as

efficient as anaerobically prepared HemW

8 with the cluster. Similiarly, the HemW

triple mutant (HemW-C16SC20SC23S) also bound heme with high efficiency. In agreement, the Mössbauer spectrum of HemW supplemented with non-enriched heme (natural isotope distribution, only 2.2 %

Fe) showed the same spectrum and identical fit parameters as HemW without further additions. The presence of heme only slightly changed the observed redox potential of the iron-sulfur-cluster of around -410 mV (Fig. 3). These results demonstrate that the iron sulfur cluster was not affected by heme binding. Vice versa, the iron sulfur cluster did not influence heme binding by HemW. Moreover the presence of SAM did not change heme binding of HemW.

Respiratory nitrate reductase and bacterioferritin are interaction partners of HemW - In order to determine specific targets for the potential heme chaperone HemW multiple interaction partners were tested using the BACTH (Bacterial Adenylate Cyclase Two-Hybrid) system in Pseudomonas aeruginosa. The P. aeruginosa system was employed because high background noise levels were observed for similar experiments in E. coli, which obscured the results. In this study, the hemoenzymes bacterial ferritin BfrA, bacterioferritin BfrB, catalase KatA, the last enzyme of heme biosynthesis ferrochelatase HemH and the heme containing subunit of the respiratory nitrate reductase NarI were analyzed for their interaction with HemW. The choices for testing BfrA and B as well KatA and Ferrochelatase HemH are obvious, since all proteins are heme binding/storing enzymes. The consecutive ß-galactosidase assays reveled the highest Miller units for the combination of HemW with NarI indicating their strong affinity.

Furthermore, HemW interacted with BfrA and BfrB but not with HemH or KatA. In agreement, in the inverse experiment

HemH and KatA neither interacted with HemW (Fig. 8).

HemW transfers heme to heme-depleted quinol-nitrate oxidoreductase NarGHI - The strongest interaction of HemW was found with the heme containing subunit NarI of the respiratory nitrate oxidoreductase NarGHI. Under anaerobic conditions and the presence of nitrate E. coli utilizes this enzyme for energy generation by replacing oxygen with nitrate as terminal electron acceptor. This respiratory complex has the ability to use all three natural quinones for energy generation (23,24). For our approach, ubiquinol served as electron donor and the membrane-anchored subunit NarI provides the quinol binding and oxidation site. Two low-spin hemes (b

and b

) involved in the electron transfer from quinols to the subunit NarG are coordinated by NarI (25). The presence of both hemes was demonstrated to be essential for the oxidation of quinols and the overall activity of NarGHI as deduced from analysis of NarI variants having lost either heme b

or b

(26). In order to unambiguously identify HemW as true heme chaperone, heme transfer analyses from HemW to the quinol nitrate oxidoreductase NarGHI from E. coli were performed. Thus, Nar-enriched membrane vesicles from E. coli wildtype MC4100 and the heme deficient E. coli ΔhemA were prepared. The absence of heme in the heme-depleted membrane vesicles due to the ΔhemA gene mutation was obvious by the visible change in color of the membrane preparation (Fig. 9B) and the corresponding UV-Vis spectra (Fig. 9A).

First, the enzymatic activity of heme-

depleted nitrate oxidoreductase was tested

spectrophotometrically using a quinol

analog as electron donor (27). A classical

spectrophotometric activity assay for the

nitrate reductase NarGHI was used. It is

based on the absorption changes of the

employed artificial electron donor the

9 menaquinol analog 2-methyl-1,4-

naphtoquinol (menadiol). The oxidation from menadiol to menadione during the reduction of nitrate to nitrite was followed spectroscopically at 260 nm. As mentioned above, only heme containing NarGHI can catalyze menadiol oxidation in the presence of nitrate. Results of the heme transfer to the NarGHI are summarized in box plots in figure 9C. Membrane vesicles prepared from E. coli wildtype MC4100 served as positive control in this experiment (Fig. 9C, lane A). Activity assays of membrane vesicles containing heme-depleted quinol nitrate oxidoreductase revealed only low residual enzymatic activity (Fig. 9C, lane B).

However, addition of HemW pre-incubated with heme lead to a significant nitrate reductase activity (Fig. 9C, lane C).

Addition of NADH further increased the observed enzyme activity (Fig. 9C, lane D). Over 50% of the potentially possible nitrate reductase acitivity was restored. Full restoration might be hampered by the possible instablility of the heme free apo enzyme compared to the holo nitrate reductase. In the negative controls, E. coli ΔhemA mutant membrane vesicles with the addition of solely heme or apo HemW exhibited residual background activity identical to the results of the heme- free nitrate reductase (Fig. 9C, lanes E and F). Similiarly, the combination of heme, HemW and NADH did not react with the used nitrate reductase substrate (Fig. 9C, lane H). SAM did not influence the heme trasnsfer reaction. To analyze the influence of the [4Fe-4S] cluster on heme transfer, a HemW triple mutant (HemW- C16SC20SC23S) lacking the cluster was tested. The activity of NarGHI was not restored by the triple mutant HemW (Fig.

9C, lane G), while the heme binding behavior of the mutant enzyme was comparable to wildtype HemW. The observed catalytic activity of the nitrate oxidoreductase in the membrane vesicles prepared from the heme-deficient

E. coli ΔhemA mutant after addition of heme-loaded HemW clearly indicates sucessful heme transfer from HemW to the heme-requiring NarI subunit. These results support the function of HemW as heme chaperone.

Slight growth phenotype of the E. coli hemW mutant – In the light of the heme chaperone function of HemW for the respiratory nitrate reductase NarGHI, growth experiments with wildtype and a hemW mutant under anaerobic, nitrate respiratory conditions with the non- fermentable carbon source glycerol were performed. Under tested growth condition the hemW mutant showed a slight, but highly reproducible growth phenotype (Fig. 1B, blue line). In the absence of nitrate almost no growth was observed.

The observed growth of the hemW mutant indicated the presence of intact nitrate reductase and a second heme inserting system supplementing for the inactivated HemW. Backup systems for essential functions were observed in E. coli for catalases, ribonucleotide reductases, and pyruvate kinases, to name a few.

Human HemW homologue RSAD1 binds heme - RSAD1 from Homo sapiens (30 % amino acid sequence identity, 50 % homology) was analyzed for heme binding via its recombinant production in E. coli, affinity purification and heme staining (Fig. 10A). Clearly, heme bound strongly to human RSAD1. Additionally, a typical absorption spectrum was also recorded for the H. sapiens RSAD1-heme complex (Fig. 10B). Alltogether, the experiments for human RSAD1 confirmed the results for bacterial HemW suggesting the ubiquitous function of HemW/RSAD1 as heme chaperone.

Discussion

HemW protein inserts heme into proteins

of different functions in respiration. Genes

encoding HemW/RSAD1 are found in the

10 genomes of almost all organisms (bacteria,

archaea, plants, animals) with the noticable exception of fungi. The presence of hemW genes nicely correlates with the utilization of heme-dependent aerobic and anaerobic respiration. Consequently, hemW is even found in organisms which employ heme taken up from the environment for this process like Lactococci (13). In contrast, strict fermentative organisms like Clostridia, deficient in heme biosynthesis and heme uptake due to the absence of classical heme-dependent respiratory processes, are also lacking HemW. How does this radical SAM protein based heme chaperone work? As shown in the model in figure 11 heme can be derived from the biosynthesis and heme import.

Interestingly, no stable complex formation between HemW and ferrochelatase (HemH), the last enzyme of the heme biosynthesis, was observed (28). However, heme from HemW was found interacting with BfrA and BfrB. In 1999, Hassett and coworkers reported that P. aeruginosa catalase A (KatA) requires bacterial ferritin A (BfrA) for full activity. They proposed that BfrA does not only store iron for the incorporation into heme, but also the necessary prosthetic heme group of KatA (29). Consequently, BfrA could function as a heme transporter between HemH, the last enzyme of heme biosynthesis and the heme- accepting protein KatA.

Recently, a radical SAM protein (ChuW) with heme degradating activity from E. coli was described. ChuW utilizes a radical-based mechanism for the heme ring opening and the methylation of the resulting open chain tetrapyrrole (30).

E. coli ChuW has an amino acid sequence identity of 28 % to E. coli HemW.

However, almost all identical amino acid residues are part of the N-terminal radical SAM element of both proteins. No common heme binding domain was detected.

Based on these observations and data of this contribution a model for HemW as depicted in figure 11 was deduced. Heme produced in heme biosynthesis gets transferred via bacterioferritin to HemW where it is covalently bound. In the presence of the [4Fe-4S] cluster HemW dimerizes, gets located to the membrane and interacts with its target NarI. Transfer of the heme requires an intact [4Fe-4S]

cluster and might involve radical chemistry. The exact mechanism of the NADH stimulation of heme transfer remains to be determined Finally, the human RSAD1 protein was found to bind heme tightly, indicating the general importance of HemW/RSAD1 enzyme family for the heme insertion into cellular proteins. Future experiments will focus on the biochemistry of the heme release from HemW to target proteins and the role of the radical chemistry in it.

Experimental Procedures Primers, strains and plasmids

The primers, strains and plasmids used in this study are listed in table S1.

Cloning, Expression and Purification of E. coli hemW

The hemW gene was PCR-

amplified from E. coli genomic DNA using

the primers hemW

-pGEX - for and

hemW

-pGEX - rev harboring a BamHI

and a XhoI-restriction site (underlined),

respectively. The PCR product of 1137 bp

was cloned into the respectively restricted

vector pGEX-6P-1 according to the

manufacturer’s instructions, yielding the

plasmid pGEX-HemW

. The vector

encoded HemW with an N-terminal

glutathione S-transferase tag (GST-tag)

and a cleavage site for PreScission

protease (GE Healthcare, München,

Germany). GST-HemW was produced in

E. coli BL21 (DE3) with the help of the

plasmid pGEX-HemW

. Cultures of

2 liters were grown aerobically in LB and

100 mg/ml ampicillin at 200 rpm and

11 37 °C. The hemW gene expression was

induced at an attenuance of 578 nm of 0.6 by the addition of 0.5 mM isopropyl-ß- D-thiogalactoside (IPTG), and cultivation was continued overnight at 17 °C and 200 rpm. Cells were harvested by centrifugation at 4000 x g for 15 min at 4 °C. All following steps occurred under strict anaerobic conditions at 20 °C. For HemW purification, cells were resuspended in 10 ml buffer 1 (140 mM NaCl, 2.7 mM KCl, 10 mM Na

HPO

, 1.8 mM KH

PO

, 5% glycerol, 1 mM DTT, pH 7.4) and cells were disrupted by a single passage through a French Press at 19,200 p.s.i.. Cell debris and insoluble proteins were removed by centrifugation for 60 min at 25000 x g and 4 °C. The soluble protein fraction was loaded onto a glutathione sepharose column (Machery-Nagel, Düren, Germany). HemW was liberated from the column by cleavage of the GST tag overnight with PreScission protease (GE Healthcare, München, Germany) according to the manufacturer’s instructions. HemW- containing fractions were eluted, pooled and concentrated by ultrafiltration using an Amicon membrane with a 30 kDa molecular mass cut-off (Merck Millipore, Billerica, USA). Protein concentrations were determined with the colorimetric assay using the Bradford reagent with bovine serum albumin as standard according to manufacturer´s instructions.

(Sigma-Aldrich, Taufkirchen, Germany) The triple mutant hemWC16SC20SC23S was constructed using the Q5

site- directed mutagenesis kit (New England Biolabs, Frankfurt, Germany) according to manufacturer’s instructions. Successful construction of mutations was confirmed by DNA sequencing of the complete hemW gene variant. Production and purification of the HemW variant was performed analogously to wildtype HemW.

Absorption Spectroscopy

UV-visible absorption spectra of HemW and HemW-heme complexes were

recorded on a Jasco V-650 spectrophotometer (Jasco, Gross-Umstadt, Germany) in buffer 1, using the same buffer as a blank. The recording wavelengths were from 250 - 600 nm.

10 mg Heme were dissolved in 1 ml 0.1 M NaOH and incubated at RT for 1h. After addition of 1 ml Tris-HCl (1 M, pH 7.6) the solution was centrifuged at 12.100 x g at RT for 10 min. The supernatant was filtered from insoluble residues and the concentration was determined at the OD

X/58.44=x * 500= x mmol/l In vitro Iron-Sulfur Cluster Analysis

The in vitro reconstitution of [Fe-S]

clusters was performed as described previously (31). After reconstitution of the [Fe-S] cluster the excess of iron and sulfide was removed by centrifugation at 12.100 x g and 4 °C and subsequent passage of the protein solution through a NAP-25 column (GE Healthcare, München, Germany) according to the manufacturer’s instructions. The iron content of purified HemW was determined according to a protocol described elsewhere (32). After denaturation of the protein with 1 M perchloric acid, bathophenanthroline was used as the chelating reagent. The sulfur content was determined as previously described (33).

Mössbauer Spectroscopy

The final HemW concentration employed for Mössbauer spectroscopy analysis of E. coli HemW was 350 µM.

Sample preparation was performed under strict anaerobic conditions. The iron-sulfur cluster of HemW was reconstituted with

57

Fe-ammonium ferric citrate. For the

sample containing HemW supplemented

with heme an equimolar ratio of heme to

HemW was added, and the mixture

incubated overnight at 20 °C. The solutions

were transferred to 350 µl Mössbauer cups

and frozen in liquid nitrogen. Mössbauer

spectra were recorded on a spectrometer

with alternating constant acceleration of

the γ-source. The minimum experimental

line width was 0.24 mms

(full width at

12 half-height). The sample temperature was

maintained constant in an Oxford Instruments Variox cryostat, whereas the

57

Co/Rh source (1.8 GBq) was kept at room temperature. Isomer shifts are quoted relative to iron metal at 300 K.

Cyclic Voltammetry

Cyclic voltammetry measurements were performed using an Ametek Versastat 3. The measurements were carried out in a self-made anaerobic three-electrode electrochemical cell flushed with nitrogen.

As the reference, a silver/silver-chloride electrode was used (3 mol l

KCl). All potentials in the text and figures are given vs. NHE (+210 mV). A platinum wire was used as the counter electrode, with glassy carbon as the working electrode. Before each measurement the platinum wire was annealed in a natural gas flame and the glassy carbon electrode was pretreated in nitric acid, neutralized, polished with 0.05 µm alumina and annealed again in a natural gas flame. For each experiment 20 cycles were recorded. The potential slightly drifted only over the first 10 cycles and stabilized thereafter. In this work, only the stabilized potential is discussed. The cycles were recorded with a scan rate of 1 V s

. All electrochemical experiments were carried out at ambient temperature in a 100 µl drop. Samples contained 120 µM HemW, 120 µM heme or 500 µM S-adenosylmethionine (Sigma-Aldrich, Taufkirchen, Germany) in diverse combinations. Samples were prepared under anaerobic conditions in a glove box (Coy Laboratories) and transferred into HPLC vials before injecting into the CV chamber directly on the glassy carbon electrode.

SAM binding and cleavage analyses The SAM-binding assays were performed as described previously (34).

For this purpose 100 µM purified HemW or BSA were incubated 0.5 µCi S-[carboxyl-

C] SAM

(1.48-2.22 GBq/mmol, 0.1 mCi/ml) at 25 °C for 1h. Mixtures were separated via

chromatography through am illustraTM NAPTM-5 desaltiung column (GE Healthcared, Freiburg, Germany).

Fractions of 200 µl were collected and analyzed by liquid scintillation counting (Perkin Elmer, Waltham, USA).

For SAM cleavage, 25 µM purified HemW (free or loaded with heme) were incubated with 0.6 mM sodium dithionite as potential electron donor and 0.6 mM SAM overnight at 17 °C under anaerobic conditions.

Reactions were stopped by adding 5 % formic acid. For HPLC analysis the samples were centrifuged at 16.100 x g für 10 min. HPLC analysis was performed as described previously (20). In detail, for the separation of 5’-deoxyadenosine from SAM a hypercarb column (Thermo Fisher Scientific, Waltham, USA) at a JASCO 2000 system (JASCO, Groß-Umstadt, Germany) with a flow rate of 0.2 ml/min was used at room teperature. A 5 ml gradient of 0.1 % TFA in H

O and 0.08 % TFA in acetonitrile was applied.

SAM and 5’-deoxyadenosine were detected at 254 nm. Appropriate markers were used to calibrate the column.

Determination of the Native Molecular Mass

An Äkta purifier system for gel permeation chromatography with a Superdex 200 HR 10/300 column was used (GE Healthcare, München, Germany). The column was equilibrated using buffer 1 and calibrated using carbonic anhydrase (Mr = 9,000), bovine serum albumin (Mr = 66,200), yeast alcohol dehydrogenase (Mr = 150,000) and β-amylase (Mr = 200,000) as marker proteins. A sample containing purified recombinant HemW was chromatographed under identical conditions with a flow rate of 0.25 ml/min under anaerobic conditions.

Heme binding assays

One mg heme was dissolved in

100 µl of 100 mM NaOH and thoroughly

mixed (34). After 30 min 100 µl 1 M Tris,

pH 7.4 was added. The solution was

13 centrifuged at 4 °C for 10 min at

12,100 x g. The concentration was determined using ε

= 58.44 (mM cm)

. HemW was incubated with an equimolar concentration of heme under anaerobic conditions at 20 °C overnight and the formed complex was used for further analyses including spectroscopy, heme staining, and the in vitro transfer reaction to NarGHI.

For detection of the stable HemW-heme complex, the heme-complexed protein was separated via SDS PAGE followed by electrophoretic transfer to an Amersham Hybond-ECL nitrocellulose membrane (GE Healthcare, München, Germany).

After three washing steps with buffer 2 (137 mM NaCl, 2.7 mM KCl, 10 mM Na

HPO

, 1.8 mM KH

PO

) the nitrocellulose membrane was incubated for 5 min with Amersham™ ECL™ Prime Western Blotting Detection Reagent (GE Healthcare, München, Germany). Heme was detected by its intrinsic peroxidase activity (35) with a CCD camera. Acidic butanone extraction was performed as described elsewhere (22,36).

Protein/Protein Interaction studies using Bacterial Adenylate Cyclase Two-hybrid system (BACTH)

The BACTH (Bacterial Adenylate Cyclase Two-hybrid) System Kit (Euromedex, Souffelweyersheim, Frankreich) was used to analyse the interaction between P. aeruginosa HemW and selected partner proteins from the same bacterium (BfrA, KatA, HemH, BfrB and NarI). Corresponding genes were amplified by PCR using P. aeruginosa genomic DNA as template DNA. Used primers were listed in table S1. Genes were integrated into plasmids pKT25, pKNT25, pUT18 and pUT18C, respectively (Euromedex, Souffelweyersheim, Frankreich). For the detection of the in vivo interaction, selected plasmids, encoding the genes for the proteins of interest, were cotransformed into the

reporter strain E. coli BTH101 cells and washed after transformation twice with M63 buffer (2g (NH

)

SO

, 13.6 g KH

PO

, 0.5 mg FeSO

·7 H

O, 1 ml 1 M MgSO

·7 H

O,

10 ml 20 % maltose, 2 ml 0.05% thiamin, pH 7.0). Colonies were selected on M63 agar plates containing ampicillin (100 µg/ml) or kanamycin (50 µg/ml) or streptomycin (100 µg/ml), 5-bromo-4- chloro-3-indolyl-β-D-galactopyranoside (X-Gal) (40 µg/ml) and IPTG (0,5 mM).

Incubation for 4 to 8 days was at 30 °C. β- Galactosidase assays were performed accordingly to the method of Miller (37).

For this purpose, blue E. coli BTH101 colonies were used to inoculate LB medium supplemented with 100 µg/ml ampicillin and 50 µg/ml kanamycin.

Hundred µl bacterial suspension were centrifuged at 4.400 x g for 5 min at 4 °C.

The resulting pellet was resuspended in 900 µl of Z buffer (60 mM Na

PO

·7 H

O, 40 mM NaH

PO

·H

O, 10 mM KCl, 1 mM MgSO

·7 H

O,

50 mM ß-mercaptoethanol) and the cell density was measured at 600 nm.

Afterwards, one drop of 0,1 % SDS and chloroform were added. The sample was incubated for 5 min at 30 °C and 300 rpm.

The reaction was started by adding 200 µl ONPG (o-Nitrophenyl-β-

D-galactopyranosid, 4 mg/ml) dissolved in

100 mM phosphate buffer (60 mM

Na

HPO

·7H

O and 40 mM

NaH

PO

·H

O, pH 7.0). The suspension

was mixed and incubated at 30 °C. The

reaction was terminated by adding 500 µl

1 M Na

CO

. A centrifugation step for

5 min at 12700 x g removed cell debris and

chloroform. Optical densities were

recorded at 420 nm and 550 nm. Miller

Units (MU) were calculated MU 1000 x

(OD

- (1.75 x OD

) / (T

x V x OD

),

where T

stands for time of the reaction in

minutes, V for volume of culture used in

the assay in ml. The units indicate the

change in A

/min/ml of cells/OD

.

14 Heme transfer to heme-free quinol nitrate

oxidoreductase NarGHI

The heme auxotroph hemA strain SHSP18 (38) was transformed with the pVA700 plasmid (39) allowing overproduction of the NarGHI complex.

Isolation of the transformants was performed on LB-agar plate supplemented with 40 mM glucose and 150 µM δ- aminolevulinic acid. The following steps were performed simultaneously for the hemA strain SHSP18 and the wild type strain E. coli MC4100 (40) both transformed with the pVA700 plasmid. An LB overnight culture supplemented with glucose (40 mM), sodium formate (12.5 mM), sodium selenite (2 µM),

sodium molybdate (2 µM) and phosphate buffer (100 mM, pH 6.8) was inoculated with a single colony of the corresponding strains. The production culture was then inoculated with this overnight culture to an initial OD

of 0.05 in the identical medium and incubated for 24 hours at 37 °C. The grown cells were pelleted by centrifugation and kept at -20 °C until use.

Cells were resuspended in 50 mM MOPS buffer, 1 mM MgCl

, pH 7.2 and broken by two passages through a French press at 1100 p.s.i. Intact cells and cell debris were removed by a centrifugation at 14000 x g.

Membrane vesicles were obtained after ultracentrifugation at 40,000 x g for 90 minutes and kept at -80 °C until use. The amount of NarGHI in the various preparations was determined via immuno- electrophoresis. The various membrane vesicle preparations from the wildtype strain had 85 to 95 mg/ml total protein with 5.6 to 7.9 mg/ml (6.5 - 8.3 %) NarGHI protein, while preparation from the hemA mutant yielded 60 to 106 mg/ml total protein with 3.6 to 5.8 mg/ml (5.5 - 6.0%) NarGHI protein. Standard deviation between 3 to 5 % were observed. All measurements were performed with equal amounts NarGHI (4 g) and 1.5 M purified HemW/HemW-C16SC20SC23S.

For the assay 20 mM 2-methyl-1,4-

naphtoquinol (menadiol) as electron donor, 5 mM NADH, 1.5 M mM free heme, 2 mM nitrate as electron acceptor were added where indicated. A quartz cell with 1.4 ml volume was used. The assay was performed under strict anaerobic conditions at 30 °C. The activity of E. coli quinol nitrate oxidoreductase was measured spectrophotometrically as outlined before (27). The changes in absorption of the ubiquinol analog 2-ethyl- 4-naphtoquinol (menadiol) caused by oxidation was measured at 260 nm. A quartz cell with 1.4 ml volume was used.

The assay was performed under strict anaerobic conditions at 30 °C. One unit of quinol nitrate oxidoreductase activity is the amount of nitrate oxidoreductase catalyzing the production of 1 µmol of menadione per min.

Acknowledgements - This work was supported by grants from the Deutsche Forschungsgemeinschaft (Forschergruppe FOR1220 PROTRAIN). We especially thank Léa Sylvi for excellent technical assistance and Gunhild Layer for critical reading of the manuscript.

Conflict of Interest - The authors declare that they have no conflicts of interest with the contents of this article.

Author Contribution - VH, SK, TM, SB,

KM, PS performed the experiments, FA

and AM prepared the NarGHI heme-

depleted membrane vesicles, EB and WL

performed the Mössbauer spectroscopy

with HemW, KK, MB provided the

Fe,

PS provided electrochemical analyses; and

VH, MJ and DJ designed the experiments

and wrote the manuscript.

15 REFERENCES

1. Frey, P. A., Hegeman, A. D., and Ruzicka, F. J. (2008) The Radical SAM Superfamily. Crit Rev Biochem Mol Biol 43, 63-88 2. Landgraf, B. J., McCarthy, E. L., and Booker, S. J. (2016) Radical S-Adenosylmethionine Enzymes in Human Health and Disease. Annu Rev Biochem 85, 485-514

3. Wang, J., Woldring, R. P., Roman-Melendez, G. D., McClain, A. M., Alzua, B. R., and Marsh, E. N. (2014) Recent advances in radical SAM enzymology: new structures and mechanisms. ACS Chem Biol 9, 1929-1938

4. Byer, A. S., Shepard, E. M., Peters, J. W., and Broderick, J.

B. (2015) Radical S-adenosyl-L- methionine chemistry in the synthesis of hydrogenase and nitrogenase metal cofactors. J Biol Chem 290, 3987-3994 5. Frey, P. A., and Reed, G. H.

(2011) Pyridoxal-5'-phosphate as the catalyst for radical isomerization in reactions of PLP-dependent aminomutases.

Biochim Biophys Acta 1814, 1548-1557

6. Mehta, A. P., Abdelwahed, S.

H., Mahanta, N., Fedoseyenko, D., Philmus, B., Cooper, L. E., Liu, Y., Jhulki, I., Ealick, S. E., and Begley, T. P. (2015) Radical S-adenosylmethionine (SAM) enzymes in cofactor biosynthesis: a treasure trove of complex organic radical rearrangement reactions. J Biol Chem 290, 3980-3986

7. Sanyal, I., Cohen, G., and Flint, D. H. (1994) Biotin synthase:

purification, characterization as a [2Fe-2S]cluster protein, and in vitro activity of the Escherichia coli bioB gene product.

Biochemistry 33, 3625-3631 8. Helbig, K. J., and Beard, M. R.

(2014) The role of viperin in the innate antiviral response. J Mol Biol 426, 1210-1219

9. Layer, G., Moser, J., Heinz, D.

W., Jahn, D., and Schubert, W.

D. (2003) Crystal structure of coproporphyrinogen III oxidase reveals cofactor geometry of Radical SAM enzymes. EMBO J 22, 6214-6224

10. Layer, G., Verfurth, K., Mahlitz, E., and Jahn, D. (2002) Oxygen- independent

coproporphyrinogen-III oxidase HemN from Escherichia coli. J Biol Chem 277, 34136-34142 11. Layer, G., Grage, K., Teschner,

T., Schunemann, V., Breckau, D., Masoumi, A., Jahn, M., Heathcote, P., Trautwein, A. X., and Jahn, D. (2005) Radical S- adenosylmethionine enzyme coproporphyrinogen III oxidase HemN: functional features of the [4Fe-4S] cluster and the two

bound S-adenosyl-L-

methionines. J Biol Chem 280, 29038-29046

12. Dailey, H. A., Gerdes, S., Dailey, T. A., Burch, J. S., and Phillips, J. D. (2015) Noncanonical coproporphyrin- dependent bacterial heme biosynthesis pathway that does not use protoporphyrin. Proc Natl Acad Sci U S A 112, 2210- 2215

13. Abicht, H. K., Martinez, J.,

Layer, G., Jahn, D., and Solioz,

M. (2012) Lactococcus lactis

HemW (HemN) is a haem-

16 binding protein with a putative

role in haem trafficking.

Biochem J 442, 335-343

14. Kranz, R. G., Richard-Fogal, C., Taylor, J. S., and Frawley, E. R.

(2009) Cytochrome c biogenesis: mechanisms for covalent modifications and trafficking of heme and for heme-iron redox control.

Microbiol Mol Biol Rev 73, 510- 528, Table of Contents

15. Shepherd, M., Heath, M. D., and Poole, R. K. (2007) NikA binds heme: a new role for an Escherichia coli periplasmic nickel-binding protein.

Biochemistry 46, 5030-5037 16. Hannibal, L., Collins, D.,

Brassard, J., Chakravarti, R., Vempati, R., Dorlet, P., Santolini, J., Dawson, J. H., and Stuehr, D. J. (2012) Heme binding properties of glyceraldehyde-3-phosphate dehydrogenase. Biochemistry 51, 8514-8529

17. Hannappel, A., Bundschuh, F.

A., and Ludwig, B. (2012) Role of Surf1 in heme recruitment for bacterial COX biogenesis.

Biochim Biophys Acta 1817, 928-937

18. Layer, G., Pierik, A. J., Trost, M., Rigby, S. E., Leech, H. K., Grage, K., Breckau, D., Astner, I., Jansch, L., Heathcote, P., Warren, M. J., Heinz, D. W., and Jahn, D. (2006) The substrate radical of Escherichia coli oxygen-independent

coproporphyrinogen III oxidase HemN. J Biol Chem 281, 15727- 15734

19. Xu, K., Delling, J., and Elliott, T.

(1992) The genes required for heme synthesis in Salmonella typhimurium include those encoding alternative functions

for aerobic and anaerobic coproporphyrinogen oxidation. J Bacteriol 174, 3953-3963

20. Kuehner, M., Schweyen, P., Hoffmann, M., Ramos, J. V., Reijerse, E. J., Lubitz, W., Broering, M., and Layer, G.

(2016) The auxiliary [4Fe-4S]

cluster of the Radical SAM

heme synthase from

Methanosarcina barkeri is involved in electron transfer.

Chemical Science 7, 4633- 4643

21. Rouault, T. A. (2015) Mammalian iron-sulphur proteins: novel insights into biogenesis and function. Nat Rev Mol Cell Biol 16, 45-55 22. Teale, F. W. (1959) Cleavage of

the haem-protein link by acid methylethylketone. Biochim Biophys Acta 35, 543

23. Rendon, J., Pilet, E., Fahs, Z., Seduk, F., Sylvi, L., Hajj Chehade, M., Pierrel, F., Guigliarelli, B., Magalon, A., and Grimaldi, S. (2015) Demethylmenaquinol is a substrate of Escherichia coli nitrate reductase A (NarGHI) and forms a stable semiquinone intermediate at the NarGHI quinol oxidation site. Biochim Biophys Acta 1847, 739-747 24. Unden, G., Steinmetz, P. A.,

and Degreif-Dunnwald, P.

(2014) The Aerobic and Anaerobic Respiratory Chain of Escherichia coli and Salmonella enterica: Enzymes and Energetics. EcoSal Plus 6 25. Bertero, M. G., Rothery, R. A.,

Palak, M., Hou, C., Lim, D.,

Blasco, F., Weiner, J. H., and

Strynadka, N. C. (2003) Insights

into the respiratory electron

transfer pathway from the

17 structure of nitrate reductase A.

Nat Struct Biol 10, 681-687 26. Magalon, A., Lemesle-Meunier,

D., Rothery, R. A., Frixon, C., Weiner, J. H., and Blasco, F.

(1997) Heme axial ligation by the highly conserved His residues in helix II of cytochrome b (NarI) of Escherichia coli nitrate reductase A. J Biol Chem 272, 25652-25658

27. Lanciano, P., Magalon, A., Bertrand, P., Guigliarelli, B., and Grimaldi, S. (2007) High-stability semiquinone intermediate in nitrate reductase A (NarGHI) from Escherichia coli is located in a quinol oxidation site close to heme bD. Biochemistry 46, 5323-5329

28. Dailey, H. A., Dailey, T. A., Gerdes, S., Jahn, D., Jahn, M., O'Brian, M. R., and Warren, M.

J. (2017) Prokaryotic Heme Biosynthesis: Multiple Pathways to a Common Essential Product.

Microbiol Mol Biol Rev 81

29. Ma, J. F., Ochsner, U. A., Klotz, M. G., Nanayakkara, V. K., Howell, M. L., Johnson, Z., Posey, J. E., Vasil, M. L., Monaco, J. J., and Hassett, D.

J. (1999) Bacterioferritin A modulates catalase A (KatA) activity and resistance to hydrogen peroxide in Pseudomonas aeruginosa. J Bacteriol 181, 3730-3742

30. LaMattina, J. W., Nix, D. B., and Lanzilotta, W. N. (2016) Radical new paradigm for heme degradation in Escherichia coli O157:H7. Proc Natl Acad Sci U S A 113, 12138-12143

31. Fluhe, L., Knappe, T. A., Gattner, M. J., Schafer, A., Burghaus, O., Linne, U., and Marahiel, M. A. (2012) The

radical SAM enzyme AlbA catalyzes thioether bond formation in subtilosin A. Nat Chem Biol 8, 350-357

32. Fish, W. W. (1988) Rapid colorimetric micromethod for the quantitation of complexed iron in biological samples. Methods Enzymol 158, 357-364

33. Beinert, H. (1983) Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Anal Biochem 131, 373-378

34. Storbeck, S., Walther, J., Muller, J., Parmar, V., Schiebel, H. M., Kemken, D., Dulcks, T., Warren, M. J., and Layer, G. (2009) The Pseudomonas aeruginosa nirE gene encodes the S-adenosyl- L-methionine-dependent

uroporphyrinogen III methyltransferase required for heme d(1) biosynthesis. FEBS J 276, 5973-5982

35. Owens, C. P., Du, J., Dawson, J. H., and Goulding, C. W.

(2012) Characterization of heme ligation properties of Rv0203, a secreted heme binding protein involved in Mycobacterium tuberculosis heme uptake.

Biochemistry 51, 1518-1531 36. Vargas, C., McEwan, A. G., and

Downie, J. A. (1993) Detection of c-type cytochromes using enhanced chemiluminescence.

Anal Biochem 209, 323-326 37. Griffith, K. L., and Wolf, R. E.,

Jr. (2002) Measuring beta- galactosidase activity in bacteria: cell growth, permeabilization, and enzyme assays in 96-well arrays.

Biochem Biophys Res Commun 290, 397-402

38. Sasarman, A., Surdeanu, M.,

Szegli, G., Horodniceanu, T.,

18 Greceanu, V., and Dumitrescu,

A. (1968) Hemin-deficient mutants of Escherichia coli K- 12. J Bacteriol 96, 570-572 39. Guigliarelli, B., Magalon, A.,

Asso, M., Bertrand, P., Frixon, C., Giordano, G., and Blasco, F.

(1996) Complete coordination of the four Fe-S centers of the beta subunit from Escherichia coli nitrate reductase. Physiological, biochemical, and EPR characterization of site-directed mutants lacking the highest or lowest potential [4Fe-4S]

clusters. Biochemistry 35, 4828- 4836

40. Peters, J. E., Thate, T. E., and Craig, N. L. (2003) Definition of the Escherichia coli MC4100 genome by use of a DNA array.

J Bacteriol 185, 2017-2021